Identification of Disease Relevant Post Translational Modifications of Proteins in Pulmonary Fibrosis as Novel Biochemical Marker Targets

Jacob Hull Kristensen

    Research output: Book/ReportPh.D. thesisResearch

    Abstract

    Idiopathic pulmonary fibrosis (IPF) is the most common interstitial lung disease and is associated with a heterogeneous occurrence of fibrosis but the cause of the disease is still unknown. There is no cure and the two most promising drug candidates (pirfenidone and nintedanib) only provide limited halt in disease progression and outcome. Biopsies are the standard tool in IPF diagnosis but they are time-consuming, highly invasive and often fail to provide a prognosis. Biopsies are also taken too late to provide an early diagnose that can alter outcome. Chest radiography and computed tomography are less invasive but also inadequate in specific diagnose. Thus new lung specific biomarkers are needed for the diagnosis and prognosis of IPF. Novel biomarkers would be best applicable if non-invasive and able to provide information not already accessible by other noninvasive tools such as spirometry, chest radiography and computed tomography. The levels of the two proteinases neutrophil elastase (NE) and matrix metalloproteinase-7 (MMP-7) are elevated in IPF in several studies. We believe that the activity of these proteases may be related to the progression of IPF. In the present work we aimed to discuss and highlight the roles of posttranslational modifications (PTMs) in the progression of IPF with special focus on proteolytic cleavage of lung proteins such as elastin. We also aimed to develop novel non-invasive elastin biomarkers that may contribute with higher sensitivity towards diagnosis of IPF. First, we developed biomarkers for NE-specific degradation of elastin. Monoclonal antibodies (mABs) were raised against immunogenic sites in the human elastin sequence. The mABs were screened for technical performance, specificity towards NE-degraded elastin and clinical relevance. The EL-NE mAB was selected as the best candidate for the quantification of NE-specific degradation of elastin and ELISA assay development was conducted resulting in the EL-NE assay. The assay was specific towards NE-degraded elastin and the EL-NE neo-epitope with limited reactivity towards intact elastin. Secondly, we developed biomarkers for matrix MMP-7 degradation of elastin. The screening and assay development was conducted using similar methodology to EL-NE. The ELM7 mAB was selected as the best candidate for the quantification of MMP-7-specific degradation of elastin. The assay was specific towards MMP-7-degraded elastin and the ELM7 neo-epitope with limited reactivity towards intact elastin. Finally, we tested the assays for clinical relevance in serum from patients diagnosed with IPF or lung cancer and healthy matched controls. Serum EL-NE- and ELM7 fragment levels were significantly elevated in IPF- and lung cancer patients compared to matched controls. In conclusion, we have developed two technically stable assays, EL-NE and ELM7, for the quantification of elastin degraded by NE and MMP-7 respectively. Both assays were protease
    specific. Initial clinical testing suggested clinical relevance of the assays in the quantification of the excessive lung extracellular remodelling occurring in pulmonary disorders, especially IPF.
    Original languageEnglish
    PublisherDepartment of Systems Biology, Technical University of Denmark
    Number of pages128
    Publication statusPublished - 2015

    Bibliographical note

    Industrial PhD Thesis

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